A considerable amount of epidemiological evidence indicates a strong association between the consumption of a diet rich in fruits and vegetables and decreased risks of cardiovascular disease and cancer. It is generally believed that the primary agents giving rise to these beneficial health effects are phytochemical antioxidants. These compounds effectively neutralize free radicals, active atoms or molecules that can damage DNA and corrode cell membranes. Free radicals play a key role in the development of a number of adverse health conditions, including cancer, cardiovascular disease, and cataracts, and have also been implicated in both initiation and acceleration of the aging process. The results of an extensive body of research indicate the existence of a variety of beneficial properties of free radical scavengers and antioxidants, including anti-mutagenic, anti-inflammatory, anti-atherosclerotic, anti-diabetic, anti-hepatotoxic, and anti-aging properties, as well as the utility of such agents in a variety of neurological disorders. Given the wide range and gravity of the adverse health conditions associated with free radicals and other inflammatory factors, there is a critical need for agents capable of exerting antioxidant and anti-inflammatory effects to improve the pharmacological treatment of conditions such as cancer, cardiovascular disease (including atherosclerosis), cataracts, rheumatic diseases, fibromyalgia, inflammatory bowel syndrome, and Alzheimer's disease as well as numerous other neurodegenerative conditions.
Important mediators in the inflammatory response include nitric oxide (NO) and prostaglandins (PGs), which are produced at elevated levels during inflammation. Nearly two decades ago, NO was found to be released by the vascular endothelium and to be a mediator of vasodilator-induced relaxation. Thus, NO is now widely known as endothelium-derived relaxing factor and the endogenous production of NO has been shown to play an essential role in physiological regulation and host defense mechanisms. In innate immunity, production of NO counters microbial and parasitic invasion and is associated with cytotoxic and cytostatic activities against bacteria and tumor cells. Additionally, increasing evidence indicates the importance of NO in the modulation of inflammation and the overproduction of NO has been found during the progress of many inflammatory diseases such as rheumatoid arthritis and osteoarthritis.
NO is a short-lived free radical produced from L-arginine in a reaction catalyzed by NO synthase (NOS). NO mediates diverse functions by acting on most cells of the body via interaction with a variety of molecular targets that are consequently either activated or inhibited [1]. At least three types of NOS isoforms have been reported [2]. Endothelial NOS and neuronal NOS are constitutively expressed and are Ca2+/calmodulin dependent, whereas expression of the high-output isoform, inducible NOS (iNOS) occurs in activated macrophages and endothelial cells following induction by cytokines such as interferon (IFN) α, β, and γ, interleukin (IL)-1α and -1β, and lipopolysaccharide (LPS) [3]. Low concentrations of NO produced by iNOS are implicated in the antimicrobial activity of macrophages against pathogens [4]. Excessive production of NO and its derivatives, such as peroxynitrite and nitrogen dioxide, has been suggested to be mutagenic in vivo, and to provoke the pathogenesis of septic shock and diverse autoimmune disorders [5-8]. Furthermore, NO and its oxidized forms have also been shown to be carcinogenic [9]. Therefore, key therapeutic targets for such adverse conditions include the reduction of NO activity by neutralization of NO as well as reduction of the generation of NO via inhibition of iNOS activity and/or inhibition of iNOS expression.
PGs are important inflammatory mediators that are synthesized from fatty acid precursors via the cyclooxygenase pathway. Two different isoforms of cyclooxygenase (COX), designated cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), have been identified. COX-1 is a constitutive isoform that exists in most tissues and is responsible for the production of prostanoids involved in homeostasis. COX-2 is undetectable in most normal tissues, but is induced by cytokines, growth factors, oncogenes and tumor promoters, and is responsible for the production of prostanoids involved in inflammation. Enhanced levels of COX-2 have been found in humans during the course of numerous inflammatory conditions including rheumatoid arthritis, osteoarthritis and acute or chronic inflammatory disease. As the induction of COX-2 is responsible for the production of PGs at the site of inflammation, this enzyme, like iNOS, represents a possible target for therapeutic purposes [10].
Although the use of herbal therapy or alternative medicine represents an increasingly attractive approach for the treatment of various inflammatory disorders, there is often little scientific data to support such uses. Accordingly, we have investigated plants and botanical extracts to identify specific compounds possessing bioactivities applicable to the treatment of inflammatory disorders and to understand the biochemical mechanisms of such activities. Anti-inflammatory properties of various phytochemicals are mediated through inhibition of the production of cytokines (IL-1β, TNF-α, IL-6, IL-12, IFN-γ), nitric oxide (NO), prostaglandins and leukotrienes [11]. Antioxidants such as (−)-epigallocatechin-3-gallate (EGCG) [12], resveratrol [13] and naturally occurring flavonoids including apigenin and kaempferol [14] have been reported to suppress NO production through inhibition of NF-κB.
Pycnanthus angolensis Warb. (P. Kombo), commonly known as African nutmeg, is a tropical plant belonging to the Myristicaceae family and having a geographical distribution stretching across western Africa from Guinea to Cameroon, including the countries of Sierra Leone, Liberia, Cote D'Ivoire, Ghana, Togo, Benin, Nigeria, Equatorial Guinea, Angola and Uganda [15]. The plant thrives well in secondary forests, growing up to 120 feet tall, and produces fruit annually, typically between September and April. The oblong-shaped fruits, about 1.5 inches long, contain oil-rich seeds encased in a hard shell. The seeds are ready for harvest between December and April. The indigenous populations have devised a variety of uses for virtually all parts of the plant, ranging from incorporation of the plant in furniture, condiments, soaps, and cattle feed to medicinal uses.
Traditional medicinal uses for the plant utilize the bark, roots, leaves, and seeds. The pounded bark is used by the Ibos of Nigeria as a mouthwash and also as a remedy for toothaches and appetite loss. Infusions of the bark are used to prevent abortions, anemia, headache, and scabies [16]. Decoctions of the bark are used as an enema to purify breast milk for nursing mothers, as a purgative, and as an antidote for poisons. Other uses include the treatment of body aches, chest pains, skin lesions, and rashes due to river blindness (Onchocercearsis) and leprosy. Root infusions containing additional herbs are used as an antihelmintic [17]. Decoctions of the leaves are used as an enema to treat toothache and prevent miscarriage. The leaf juice is sucked to cure white tongue thrush and the latex is applied to wounds as an anticoagulant [17]. The Ewes in Ghana use fat from the oil-rich seeds as a mouthwash to cure thrush and as a topical treatment for fungal skin infections. In addition to such medicinal uses, the seeds provide an important source of oil and wax in the local communities for making candles, soap, fuel and lubricants. The oil residue is used as manure and cattle feed, and there are also reports of its use as a condiment in Equatorial Guinea.
Relatively few studies have investigated the chemical components of Pycnanthus angolensis. Two terpenoid-type quinines (pycnanthoquinone A and B) with antihyperglycemic activity have been isolated from the leaves and stem extracts [18,19] and the lignan dihydroguaiaretic acid has been identified in a bark extract [20]. Two isoflavones, 2′-hydroxyformononetin and 7,4′-dimethoxy-2′-hydroxyisoflavone, have been identified in the heartwood of the plant [21]. The seed fat is characterized by the presence of relatively high contents of tetradecanoic acid (60%) and (Z)-9-tetradecenoic acid (20%) as well as other unsaturated fatty acids [22,23,24]. A recent study of the seed fat identified a novel polyphenylated hydroquinone carboxylic acid named kombic acid [24], although the structure of this compound as reported there is questionable based on our own research findings. We have discovered that kombo butter, kombo butter acid extract, and three compounds, sargaquinoic acid, sargachromenol, and sargahydroquinoic acid, from the seeds of African nutmeg possess activity as antioxidants, NO scavengers, and inhibitors of the production of NO, iNOS, and COX-2, and are thus useful for the prevention and treatment of a number of adverse health conditions linked to inflammation in human, animal, and avian subjects.
The anti-inflammatory and antioxidant activities of sargachromenol and sargahydroquinoic acid isolated from Roldana barba-johannis have been evaluated [25]. The anti-inflammatory activity of sargachromenol exceeded that of α-tocopherol, and that of sargahydroquinoic acid was on par with that of a-tocopherol as measured by the TPA-induced mouse ear edema assay. Both of these compounds had less anti-inflammatory activity than indomethacin. The antioxidant activities of these compounds, as measured by the DPPH radical scavenging assay, were found to be better than those of either α-tocopherol or indomethacin. The authors noted that the anti-inflammatory activities of sargachromenol and sargahydroquinoic acid could be related both to their ability to scavenge free radicals and to their possible interaction with enzymes that catalyze the formation of intermediaries in the inflammatory process.
U.S. Pat. No. 6,489,494 to Leonard discloses antioxidants comprising kombic acid, a substituted palmitic fatty acid, or derivatives thereof obtained from crude kombo butter, as well as methods for the isolation of such antioxidants from crude kombo butter. Leonard found that the solubility of kombic acid in either alcohol or supercritical carbon dioxide was much less than that of more common fatty acids and glycerides, and thus that extraction with either of these solvents represented a commercially viable means for the isolation of kombic acid from kombo butter. Leonard also showed that the antioxidant activity of kombic acid with respect to lipid peroxidation either met or exceeded that of α-tocopherol in in vitro assays, and that kombic acid was an effective antioxidant for the stabilization of edible oils, plastics, and cosmetics. U.S. Pat. No. 6,713,512 to Leonard, a continuation-in-part of the '494 patent, discloses the use of kombic acid and derivatives thereof as anticancer and cholesterol-lowering agents.
Published United States Patent Application No. 20030129294A1 to Barclay et al. discloses blends comprising triglycerides and a hydroquinone substituted polyunsaturated fatty acid, as present in kombo butter, or a derivative thereof. A particularly preferred substituted fatty acid is sargahydroquinoic acid. Additionally, the blends may comprise glycerides from kombo butter and/or antioxidant compounds to stabilize the acids of the invention. Also disclosed are food products and dietary supplements having a health component that is a hydroquinone substituted polyunsaturated fatty acid as present in kombo butter, extracts thereof, or a derivative thereof, and particularly sargahydroquinoic acid. Barclay discloses the activity of sargahydroquinoic acid as an inhibitor of pancreatic lipase, a stimulant for increased production of the dermal protein decorin, a ligand for peroxisome proliferator activated receptor (PPAR) subtypes α and γ, an inhibitor of cholesterol esterase and acetylcholine esterase, and an inhibitor of microbial viability and fatty acid catabolism. In light of these findings, Barclay postulates the utility of the hydroquinone substituted polyunsaturated fatty acid preparations of the invention in: treatment and prevention of obesity (pancreatic/gastric lipase inhibitor); improved lipid metabolism, control of hyperlipidemia, and treatment and prevention of cancer (PPAR-α ligand); control of insulin sensitivity to avert short-term problems of insulin resistance including cognitive impairment (poor memory), chronic fatigue, and mood swings, as well as long-term problems including cardiovascular disease, type-2 diabetes, and polycystic ovary syndrome (PPAR-γ ligand); improved cognition, improved regulation of blood sugar levels, cellular differentiation, lipid metabolism, and glucose metabolism, and treatment and prevention of inflammation, cancer, gestational diabetes, syndrome X, hypertension, and stroke (also as a PPAR-γ ligand); prevention of aging of the skin due to stimulation of the production of the dermal proteins decorin and collagen; and decreased oral and body odor due to the inhibition of microbial viability and fatty acid catabolism.
Additionally, Barclay discloses the antioxidant activity of a kombo nut extract containing approximately 56% sargahydroquinoic acid. Incorporation of 0.5% of the extract in sunflower oil was found to extend the swift induction period for oxidative degradation of the oil from 3 to 17 hours, while incorporation of the same level of a 50% preparation of mixed tocopherols provided only a 4-hour induction period. Barclay also discloses that kombo butter saponifiables exhibited a dose-dependent reduction in levels of prostaglandin E2 in human skin fibroblasts after induction with phorbol myristate acetate in vitro. In light of these findings, Barclay postulates the utility of the hydroquinone substituted polyunsaturated fatty acid preparations of the invention in the treatment and prevention of disease conditions relating to inflammation and the presence of free radicals, including cardiovascular disease, joint diseases, arthritis, peptic ulcer disease, inflammatory bowel disease, inflammatory skin conditions, neurodegenerative diseases (including Alzheimer's), and allergies.
Finally, Barclay discloses a process to enrich a hydroquinone substituted polyunsaturated fatty acid from kombo butter. According to the process, kombo butter is refined by the addition of base at a temperature of 40 to 60° C., and the product is separated into organic and aqueous phases. The aqueous phase is acidified to a pH of 0 to 4, and the oily layer is drained off. This affords recovery of a product containing more than 20% by weight of the hydroquinone substituted polyunsaturated fatty acid.
Published United States Patent Application No. 20030206936A1 to Barclay et al. discloses cosmetic compositions comprising an effective amount of a hydroxy phenyl alkyl carboxylic acid, preferably sargahydroquinoic acid or a derivative thereof. The cosmetic benefits disclosed for such active compounds include: treating and preventing wrinkling, sagging, aging and/or photodamaging of skin; boosting the production of collagen and decorin in the skin; soothing irritated, red and/or sensitive skin; improving skin or scalp texture, smoothness, and firmness; reducing body odor; reducing or preventing dandruff, spots, and pimples; and lightening skin or preventing the darkening of skin. The use of kombo butter, its partial glycerides, or its free fatty acids to obtain these cosmetic health benefits is also disclosed. Mirroring the disclosure of the previous Barclay application (No. US20030129294A1) discussed above, examples are presented indicating the utility of sargahydroquinoic acid as a stimulant for increased production of the dermal protein decorin and as a ligand for PPAR subtypes α and γ, as well as the dose-dependent reduction of prostaglandin E2 (PGE2) levels by kombo butter saponifiables in human skin fibroblasts in vitro.
Published United States Patent Application No. 20030181521A1 to Leonard et al. discloses the use of cetyl myristoleate from kombo butter for the treatment of osteoarthritis and orthopedic or muscular injuries in equines. Leonard found that kombo butter-based cetyl myristoleate preparations were much more palatable to horses than preparations derived from beef tallow, thus making ingestion a feasible route of administration for the compound.
Despite the aforementioned references, nothing in the prior art discloses the ability of kombo butter, kombo butter acid extract, sargaquinoic acid, sargachromenol, or sargahydroquinoic acid to inhibit the production of NO and the expression of iNOS protein and/or mRNA. Moreover, although the Barclay applications disclose the ability of kombo butter saponifiables to reduce the levels of PGE2 in human skin fibroblasts in vitro, the use of kombo butter, kombo butter acid extract, sargachromenol, and sargahydroquinoic acid to reduce COX-2 protein and/or mRNA expression is unknown in the prior art. Thus, we have discovered that kombo butter, kombo butter acid extract, sargaquinoic acid, sargachromenol, and sargahydroquinoic acid are useful in reducing NO levels during inflammation not only by radical scavenging, but also advantageously by acting to reduce the levels of NO production in response to inflammatory stimuli at the levels of transcription and translation of iNOS. We have also discovered that kombo butter, sargachromenol, and sargahydroquinoic acid effectively inhibit both COX-2 protein and mRNA expression and that kombo butter acid extract also inhibits expression of the COX-2 enzyme.